Note: Descriptions are shown in the official language in which they were submitted.
W094/29439 ~ PCT~S94/06S41
.
HIG~ YIELDING INFLUENZA VIRU8E8
1. Introduction
The present invention relates to influenza virus
strains which may reliably be propagated to high titer
in hosts such as embryonated eggs. High yield
strains, including reassortment influenza viruses and
influenza viruses exhibiting chimeric viral surface
molecules, such as chimeric HA and/or NA surface
molecules, are described. Further, methods for the
production of such high yield influenza A, B, and/or C
virus strains are described. These high yield viruses
may be used in the production of anti-viral vaccines
and the manufacture of chimeric influenza viruses.
2. Background Of The Invention
Anti-viral vaccine production presents many
challenges. For example, an ever-changing group of
influenza virus subtypes, such as, say, influenza
A(H3N2) and A(HlN1), and influenza B, circulate within
the general populati~n. Further, due to antigenic
drift, a large amount of variation quickly develops
within each influenza A subtype and within influenza
B. Thus, it is neceCcAry to continually isolate
influenza viruses and Acc~F5 their antigenic
characteristics. Annually, then, such information is
used in determining the composition of the influenza
vaccine for the following reason (Robertson, J.S., et
al. 1988, in Influenza Vaccines, Development and
Perspectives, Giornale di Igiene e Medicina Preventiva
29:4 58).
Strains of influenza virus, newly isolated from
patients, however, do not usually grow to high titers.
This is especially true when the viruses are
propagated in chick embryonated eggs, the preferred
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W094/29~9 PCT~S94/06541
hosts for the production of large quantities of virus
for use as killed virus vaccines.
In order to improve the growth characteristics of
newly isolated influenza virus strains, attempts are
often made to reassort the new strain with a high
growth laboratory strain. For example, new influenza
A virus strains are crossed with the high-yield
influenza A/PR/8/34 virus strain to produce
potentially high yield reassortment strains. Those
reassortment viruses with high growth characteristics
usually derive six genomic RNA segments from the high-
yield parental virus strain and the hemagglutinin (HA)
and neuraminidase (NA) segments from the newly
,5 isolated parental strain (Kilbourne, E.D. and Murphy,
J.S., 1960, J. Exp. Med. 111:387; Kilbourne, E.D.,
1969, WH0 Bull. 41:643; Baez, M. et al., 1980, J.
Infectious Dis. 141:362; Robertson, J.S. et al., 1992,
Biological 20:213). Although this A/PR/8/34 strain
reassortment approach may give some influenza virus
strains with improved growth characteristics, many of
the resulting reassortment strains will exhibit
limited growth potential in embryonated eggs.
Current methods, therefore, for increasing the
replication titer of newly discovered viral strains
are not optimal, and improved methods for increasing
viral yields in the preferred hosts, for vaccine
production would be highly beneficial for the process
of anti-viral vaccine development and manufacture.
3. SummarY Of The Invention
The present invention relates to influenza
strains which may consistently and reliably be
propagated to high titer in hosts which include, but
are not limited to embryonated eggs and embryo-derived
tissue culture cells, and may be used in the
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W094/29~9 ~ 6 ~ 9 ~ ~ PCT~Sg~/06541
production of anti-viral vaccines. The invention
further relates to methods for the production of such
high yield viruses. More specifically, the high-yield
influenza viruses of the invention contain portions of
viruses which are virulent in Mx hosts, (i.e., hosts
cont~; ni ng the Mx allele). The invention is based in
part, on the surprising finding that Mx mouse
resistant influenza virus strains grow to higher
titers in embryonated eggs than do specifically egg-
adap~ed viral strains.
The methods presented for the production of high
yield viral strains include, first, the production of
reassortment strains developed by crossing a low titer
virus strain with a virus resistant to (i.e., virulent
in) an Mx host. Second, methods for the production of
high yield virus strains are presented wherein high
yield viruses are constructed which exhibit chimeric
viral surface molecules, such as chimeric HA and/or NA
surface molecules. The chimeric surface molecules
contain the cytoplasmic or cytoplasmic and
transmembrane portions of the HA or NA molecules of a
viral strain in an Mx host, and at least the
extracellular antigenic portion of the HA or NA
molecule from the newly isolated low titer virus
strain. These methods may be utilized for the
production of high yield influenza A, B, or C virus
strains.
4. Brief Description Of The Fi~ures
FIG. 1. Diagram depicting reassortment between a
high yield Mx resistant parental strain (non-hatched)
and a recently isolated, low yield virus isolate
(hatched). The resulting reassortment virus shown
contains HA and NA surface proteins derived from the
W094/29~9 PCT~S94/06541
2~94~ ~
low yield parent and the rest of the viral components
derived from the high yield viral parent.
FIG. 2. Diagram depicting the interaction of
viral surface proteins (HA and NA) with the core
structure of influenza viruses.
FIG. 3. Diagram depicting the construction of a
high yield virus exhibiting a chimeric HA surface
protein. The two viruses shown contain extracellular
or extracellular and transmembrane domains derived
from a low yield viral strain (hatched) while the
remaining components of the virus are derived from a
high yield strain developed in a Mx host strain.
5. Detailed Description Of The Invention
The present invention is based, in part, on the
surprising discovery that viral stains developed in Mx
mice grow to higher titers in embryonated eggs than do
specifically egg-adapted viral strains. Lindenmann
(Lindenmann, J. et al., 1963, J. Immunol. 90:942)
discovered that the A2G strain of mice is resistant to
influenza A and B viruses. The resistance was later
found to be associated with the presence of the
dominant Mx allele present in several mouse lines,
including the A2G strain. Only a few influenza
viruses have been reported to be virulent in this
strain of mice (Haller, O., 1981, in "Current Topics
in Microbiology and Immunology 92, Henle, W. et al.,
Springer-Verlag: Berlin, pp. 25-51). As shown in the
Working Example presented below in Section 6,
influenza strains which are virulent in Mx-containing
A2G mice surprisingly behave as high yield viruses
when propagated in embryonated egg hosts.
W094/29~9 ~ PCT~S94/06541
Therefore, as discussed further below, instead of
the A/PR/8/34 influenza strain currently used for
generating potentially high yield reassortment
strains, viruses which are virulent in Mx hosts are
utilized as parent strains for the high yield
reassortment virus strains of the invention. Further,
as described in detail below, portions of the viral
surface proteins, such as HA and/or NA proteins,
derived from viral strains developed in Mx hosts are
utilized as part of the chimeric high yield virus
strains of the invention. Also described below are
methods for the production of the high yield
reassortment and chimeric virus strains of the
invention.
5.l Hiqh Yield Viruses
The high yield influenza viruses of the invention
are capable of growing to high titer in hosts which
include, but are not limited to such hosts as
embryonated eggs, preferably embryonated hens eggs,
and tissue culture cells derived from embryos such as
chick embryos. In addition, the high yield viruses of
the invention may be capable of growing to high titer
in a large number of other cells, including, but not
limited to mammalian and avian cell lines. At least a
portion of each high yield influenza virus is derived
from components of viral strains which have been
developed in hosts which contain and express the
dominant Mx allele. Such Mx hosts are preferably Mx
mice or Mx mouse cell lines. Additionally, Mx hosts
may include, but are not limited to any mammalian
animal, ferret for example, any mammalian cell line,
any avian animal, or avian cell line, which contains
35 and expresses the Mx allele. The Mx allele in these
appropriate hosts may be endogenous to the host or
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W094/29~9 PCT~S94/06541
~6~g
may, alternatively, be introduced into the host using
st~nA~rd recombinant DNA techni ques well known to
those of ordinary skill in the art. The high yield
influenza viruses of the invention may include, but
are not limited to, reassortment viruses and viruses
exhibiting chimeric viral surface proteins, preferably
chimeric HA and/or NA proteins.
The high yield reassortment influenza viruses of
the invention grow to high titer in both Mx hosts and
in embryonated egg and embryo-derived tissue culture
cell hosts. The high yield reassortment viruses
should exhibit on their surfaces viral surface
proteins, preferably HA and/or NA proteins, derived
from the low titer parent because these proteins are
major targets of the host immune response after
infection. The reassortment viruses should contain,
therefore, at least the gene(s) derived from the low
titer viral parent encoding one or more viral surface
proteins, such as the HA and/or NA surface proteins.
Further, the high yield reassortment viruses of the
invention include and express at least those portions
of the genome of the Mx host-derived strain which
allow the strain to grow to high titer in the Mx host.
Such portions of the viral genome may include, but are
not limited to, all or any of the genes encoding the
components of the RNA-directed-RNA polymerase complex
(PB1, PB2, and PA), the gene encoding the
nucleoprotein (NP), which forms the nucleocapsid, the
genes encoding the matrix proteins (M1, M2), and/or
the genes encoding the nonstructural proteins (NS1,
NS2). The portion of the Mx host-derived parental
strain's genome contained within the high yield
reassortment virus of the invention may, but is not
required to, induce a lower host interferon response
than the host interferon response elicited by the low
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W094/29~9 2 1 ~ ~ 9 ~ 6 PCT~S94/06541
titer viral parent, or viruses currently used to
prepare high yield reassortment.
The high yield viruses of the invention may
include viruses which exhibit chimeric viral surface
proteins, such as chimeric HA and/or NA surface
proteins. Because the core structures of influenza
viruses are thought to interact with the cytoplasmic
domains of the surface proteins, such as the HA and NA
surface proteins, chimeric viral surface proteins
cont~;n;ng cytoplasmic domains derived from a high
yield Mx resistant strain may provide more favorable
protein-protein interactions with a core structure
derived from a Mx resistant virus strain than
unaltered viral surface proteins would, thus providing
a higher growth potential (See FIG. 2 for a diagram
depicting this concept). At least the extracellular
portions of the chimeric viral surface proteins should
be derived from the extracellular portions of the
viral surface proteins of the low titer strain. The
cytoplasmic or the cytoplasmic and transmembrane
regions of the chimeric viral surface proteins should
be derived from the viral surface proteins of a viral
strain which was developed in the Mx host.
Accordingly, the portions of the viral genome encoding
the extracellular domain(s) of the chimeric viral
surface proteins should be derived from the genome of
the low titer viral strain, and the portions of the
viral genome enco~;~g cytoplasmic or cytoplasmic and
transmembrane domains of the chimeric viral surface
proteins should be derived from the genome of a viral
strain which was developed in a Mx host. In one
embodiment, the high yield influenza viruses of the
invention may consist of a virus derived almost
exclusively from a viral strain which was originally
developed in a Mx mouse strain, and contains only the
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W094/29~9 PCT~S94/06541
extracellular domain of a viral surface protein, such
as HA and/or NA, derived from a low titer viral
strain.
Portions of the low titer strain-derived domains
of the chimeric viral surface proteins of the chimeric
viruses of the invention may be modified such that the
chimeric virus strain is capable of growing to a
higher titer in hosts such as embryonated eggs. The
~O portions of the low titer strain-derived domains which
may be modified include, but are not limited to the
extracellular domain's receptor-binding sites and/or
those domains which affect the cleavability of the
surface proteins. Modifications may include, but are
not limited to amino acid insertions, deletions, or
substitutions. In all cases, the modifications do not
effectively change the antigenicity of the virus
strains of the invention which is important for a
protective host immune response.
Amino acid substitutions may be of a conserved or
non-conserved nature. Conserved amino acid
substitutions consist of replacing one or more amino
acids of the low titer strain-derived domains of the
viral surface proteins with amino acids of similar
charge, size, and/or hydrophobicity characteristics,
such as, for example, a glutamic acid (E) to aspartic
acid (D) amino acid substitution. Non-conserved
substitutions consist of replacing one or more amino
acids of the low titer strain-derived domains of the
viral surface proteins with amino acids possessing
dissimilar charge, size, and/or hydrophobicity
characteristics, such as, for example, a glutamic acid
(E) to valine (V) substitution.
Amino acid insertions may consist of single amino
acid residues or stretches of residues ranging from 2
to 15 amino acids in length. One or more insertions
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W094/29~9 ~ PCT~S94/06541
= . ~ ......... .... .
may be introduced into the low titer strain-derived
viral surface proteins so long as the insertions do
not effectively change the antigenicity of the virus
strain which is important for a protective immune
response.
Deletions of portions of the low titer strain-
derived viral surface proteins, are also within the
scope of the invention. Such deletions consist of the
removal of one or more amino acids from the protein-
sequence, so long as the resulting deletion protein
does not exhibit an effectively changed antigenicity
which does not elicit a protective immune response
from the host. Deletions may involve a single
contiguous or greater than one discrete portion of the
peptide sequences.
The high yield influenza viruses of the invention
may be used as anti-viral vaccines. Such vaccines may
be u ed in human as well as non-human hosts,
including, but not limited to equine, porcine, and
avian hosts. Vaccine administration is performed
according to te~hn;ques well known to those of
ordinary skill in the art. tSee Palese, P. et al.,
1992, U.S. Patent No. 5,166,057, which is incorporated
herein by reference in its entirety, for such
t~c~n;ques.)
5.2 Production Of Hiqh Yield Virus
Presented below are methods for the production of
the high yield viruses of the invention. Included are
methods for the development and production of high
yield reassortment viruses and high yield viruses
which exhibit chimeric viral surface proteins.
W094l29~9 PCT~S94/06541
5.2.1 Production Of Hiqh Yield Reassortment Viruses
In order to produce a high yield reassortment
virus strain, a low titer parent influenza strain is
crossed, in a standard mixed infection, with a parent
virus strain developed in a Mx host. Mixed infections
are performed by coinfecting Mx resistant virus with
various dilutions of a low titer virus strain. For
influenza A, high titer virus strains may include, but
are not limited to the PR8 strain (Haller, O., 1981,
in "Current Topics in Microbiology and Immunology",
Henle, W. et al., eds., Springer-Verlag: New York, pp.
25-51). Other high yield influenza A, B, or C
parental strains may be developed by passage though Mx
hosts and by selecting those strains which exhibit
virulence and high growth characteristics in Mx hosts.
Mx hosts may include, but are not limited to Mx mouse
cell lines such as the A2G cell line, and Mx mouse
strains, such as the A2G mouse strain (Lindenmann, J.
et al., 1963, J. Immunol. 90:942-951). Additionally,
Mx hosts may include, but are not limited to any
mammalian animal, ferret, for example, mammalian cell
line, any avian animal, or avian cell line which
contains and expresses the Mx allele. The Mx allele
in these appropriate hosts may be endogenous to the
host or may, alternatively, be introduced in the host
using stAn~Ard recombinant DNA techn; ques well known
to those of ordinary skill in the art. Animals or
monolayers of cells may be coinfected using standard
t~r.hn; ques well known to those in the art. In the
case of whole animal infections, hosts may, for
example, be coinfected by intranasal or aerosol
routes.
Progeny reassortment viruses resulting from mixed
infections may be passaged through Mx hosts or may be
used to infect hosts such as embryonated egg hosts or
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W094/29439 ~ ~3~ 4 PCT~S94/06541
.
embryo-derived cell hosts. The growth characteristics
of the reassortment virus strains are then evaluated
and those exhibiting high titer growth capabilities
may act as reassortment viruses of the invention.
Due to the fact that the viral surface proteins
are major targets of the host humoral response after
infection, the high yield reassortment virus strains
of the invention must exhibit on their viral surfaces
at least one viral surface protein, such as HA or NA,
which has been derived from the low titer parental
viral strain (See FIG. 1 for a diagram depicting one
embodiment of the reassortment virus strains of the
invention). One may determine whether viral surface
proteins of the reassortment strains are derived from
the low titer parental strain by, for example assaying
for their presence using antibodies raised against low
titer viral surface proteins which do not cross react
with the viral surface proteins of the high yield
parental strain. Monoclonal or polyclonal antibody
preparations may be used for these procedures.
Alternatively, one may assay for the absence of HA or
NA proteins which have been derived from the high
yield parental strains by contacting the reassortment
virus or viral proteins with antibodies raised against
the high titer parental strain-derived viral surface
proteins which do not cross react with the viral
surface proteins of the low yield parental strain.
In addition, one may assay for the presence of
the genes enco~ing low titer-derived viral proteins by
using a standard reverse transcriptase (RT):polymerase
chain reaction (PCR; the experimental embodiment set
forth in Mullis, K.B., 1987, U.S. Patent No.
4,683,202) based t~chn;que. In this instance, pairs
of primers may be used which specifically hybridize to
nucleotide sequences present in the low titer-derived
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W094/2g439 PCT~S94/06541
viral surface protein genes but do not hybridize to
sequences present in the high yield parental virus
strain-derived viral surface protein genes. PCR
amplification using st~n~Ard te~-hniques (Innis, M.A.
et al., eds., l99O, PCR Protocols, Academic Press,
NY), therefore, will only yield an amplified fragment
when RNA from a reassortment strain con~;n;ng low
titer-derived viral surface protein genes is used as
the starting material in the assay. Alternatively,
one may assay for the absence of genes encoding viral
surface proteins which have been derived from the high
yield parental strains by utilizing primer pairs which
specifically hybridize to nucleotide sequences present
in the high yield parental strain-derived viral
surface protein genes but do not hybridize to the
viral surface protein genes of the low titer strain.
No amplification will occur when RNA from those
reassortment strains which do not contain high yield
parental strain derived HA or NA genes is used as the
starting material for the assay.
When developing reassortment strains, one may
select against the presence of high yield parental
strain derived viral surface proteins, instead of
relying on completely random reassortment of RNA gene
segments. For example, progeny viruses obtained after
an initial mixed infection may be pretreated with an
antiserum raised against the viral surface proteins of
the high yield parental strain. Such treatment will
neutralize those viruses exhibiting the high yield
parental strain derived surface proteins, inhibiting
their ability to infect host cells. The antiserum-
treated virus sample, which now contains a population
of infectious viruses which are enriched for viruses
that exhibit low yield parental strain-derived viral
surface proteins, may then be used to infect
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W094/29~9 2 ~ PCT~S94/06S41
.
appropriate host cells. This is an especially useful
techn i que when utilized in the construction of the
viruses of the invention, in that neutralizing
antibodies against surface proteins may readily be
made.
The resulting reassortment virus strain may then
be used to infect hosts such as embryonated egg or
embryo-derived cell hosts, at which time growth
characteristics of the reassortment strain in the host
may be evaluated. Such growth characteristics may be
evaluated by determining, for example, the number of
PFUs per volume of viral sample or by assaying the
hemagglutinin titer of the viral sample, using
t~chn; ques well known to those of ordinary skill in
the art.
5.2.2 Production of High Yield Chimeric
Reassortment Viruses
The production of the chimeric high yield viruses
of the invention, described above in Section 5.1 is
described here. The steps involved in the production
of such chimeric viruses consists of, first, the
construction of an RNA segment containing a gene
~ncoA;ng a chimeric viral surface protein, and,
second, ribonucleoprotein (RNP) transfection to
construct the high yield strain.
In order to construct the segment containing the
chimeric viral gene, the RNA segments encoding the
viral surface protein(s) of interest (such as the HA
and/or NA proteins) from both a high yield strain
which was originally selected in an Mx host, and from
a low yield strain must be obtained. Procedures for
obtaining these RNA segments utilize standard
35 t~chn; ques well known to those of ordinary skill in
the art. See, for example, Palese and Schulman, 1976,
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W094/29439 2 1 ~ ~9 ~ ~ PCT~S94/06541
J. Virol. 17:876-884, which is incorporated herein by
reference in its entirety.
The RNA segment encoding these genes is then
reverse-transcribed into cDNA, using ~echniques well
known to those of skill in the art (Sambrook, et al.,
1989, Molecular Cloning: A Laboratory Manual, Vols.
1-3, Cold Spring Harbor Press, NY). once the genes
are present in a DNA form, the appropriate portions of
each gene may be isolated and combined using stAn~rd
recombinant DNA techniques. Using the HA viral
surface protein gene as an example, the region of the
HA gene which encodes the extracellular domain of the
HA protein of a low yield strain, may be isolated by,
for example, the use of a convenient restriction
enzyme site endogenous to the sequence or engineered
into the sequence by recombinant means (see, for
example, Kunkel, 1985, Proc. Natl. Acad. Sci. USA
82:488), or by other enzymatic or chemical means.
Next, the region of the HA gene encoding the
cytoplasmic and transmembrane domains of the HA
protein may be isolated using the same types of
procedures. The region of cDNA encoding the
extracellular domain of the low yield strain and the
cDNA encoding the cytoplasmic and transmembrane
domains of the high yield strain may then be joined,
using st~n~rd enzymatic means. Alternatively, once
the nucleotide sequence of the HA gene of the low and
high yield strains is known, the chimeric viral genes
of the invention may be synthesized using chemical
means well known to those of skill in the art.
In addition to the appropriate amino acid coding
regions, the chimeric gene constructs should include
those sequences, such as viral polymerase binding
site/promoter, RNA polymerase-binding sites, and the
appropriate 3' and 5' regulatory sequences well known
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W094/29~9 ~ PCT~S94/06541
to those of skill in the art which will allow
efficient transcription of the chimeric RNA segments.
cDNA chimeric constructs may then be introduced into a
system whereby the cDNA may be transcribed into RNA of
a negative polarity. Such systems are well known to
those of skill in the art and include, but are not
limited to the T3, T7, and SP6 in vitro transcription
systems and the like. RNA chimeric constructs may be
utilized directly.
The negative polarity RNA encoding the chimeric
viral surface NA proteins may then be combined with
viral RNA polymerase complex, which may be isolated or
may be produced using recombinant techniques, both of
which are well known to those of skill in the art, to
form infectious RNP (ribonucleoprotein) complex. RNPs
comprise viral RNA-directed RNA polymerase proteins
(the P proteins) and nucleoprotein (NP). The RNPs may
then be used together with a "parent" virus to
coinfect host cells. Appropriate host cells may
include, but are not limited to Mx mouse cells, such
as those of the mouse A2G cell line, or,
alternatively, embryonated egg or embryo-derived host
cells. The "parent" in this instance, is a high yield
virus strain originally developed in a Mx strain. The
t~chniques utilized for the production of infectious
RNPs containing genes encoding chimeric proteins may
be found in Palese et al. (Palese, P. et al., 1992,
U.S. Patent No. 5,166,057). After coinfection,
techniques essentially identical to those described
above in Section 5.2.1 may be utilized to select for
and characterize those progeny viruses which exhibit
chimeric viral surface proteins and whose remaining
components are derived from the original high yield MX
35 resistant "parental" strain.
W094/29~9 PCT~S94/06541
6. Example: Production and Characterization
Of A High Yield Reassortment
Influenza Virus
In this example, a reassortment influenza virus
developed in a Mx mouse host is described which grows
to high titer in embryonated egg hosts.
6.1 Materials And Methods
Viruses: The high yield Mx resistant parent
virus used was the PR8 virus strain (Haller, O., 1981,
in "Current Topics in Microbiology and Immunology;
Herle, W. et al., eds., Springer-Verlag: New York, pp.
25-51). A recent influenza A virus isolate was used
as the low titer parent virus strain.
Host Strains: The A2G Mx mouse strain
(Lindenmann, J. et al., 1963, J. Immunol. 90:942-951)
and eleven day old embryonated hen's eggs were used as
host strains.
Reassortment and characterization Procedures:
St~nAArd mixed infection procedures and hemagglutinin
titer assays were utilized (Palese and Schulman, 1976,
J. Virol. 17:876-884).
6.2 ~esults
Bxperiments were performed to develop a high-
yielding reassortment influenza A virus strain which
may be utilized for the production of large amounts of
virus suitable for vaccine production. To that end, a
recently isolated influenza A strain which grows to
only low titer in embryonated egg hosts and the
influenza A virus strain, PR8, a virus strain which
exhibits high titer growth in Mx mice, were used as
the parent virus strains for the production of a
reassortment virus strain. The low and high titer
parent virus strains were coinfected into embryonated
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.
eggs in a standard mixed infection procedure. Progeny
reassortment viruses produced from the coinfection
were subsequently repA-ecAged in embryonated egg host
cells, and reassortment viruses were isolated using an
- antibody screen directed against the HA and NA surface
proteins of the PR8 parent strain.
Presented in Table I, below, are results which
demonstrate that the reassortment virus produced in
this study grows to high titer in embryonated egg
hosts. The growth characteristics of the virus
strains were assayed using stAn~Ard hemagglutinin
titer measurements. Thus, the recently isolated low
yield influenza A virus was successfully used together
with a high yield PR8 virus to produce a reassortment
strain which, surprisingly, acts as a high yield virus
strain in an embryonated egg host.
TABLE 1
INFEUENZA A VIRU8 8TRAIN HE~ VTINATION (HA) TITER
Mx resistant strain 16,584
Recent isolate 128
Reassortment strain contA; n; ng HA 2,048 - 4,096
and NA of recent isolate and
remaining genes derived from
Mx resistant strain
It is apparent that many modifications and
variations of this invention as set forth here may be
made without departing from the spirit and scope
thereof. The specific embodiments described
hereinabove are given by way of example only and the
invention is limited only by the terms of the appended
claims.
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